Abstract Selenium is an essential trace element that is incorporated into 25 human proteins as the amino acid selenocysteine (Sec). The proteins that contain Sec (selenoproteins) are essential for many cellular functions including combatting oxidative stress, thyroid hormone production and protein folding. Sec is incorporated at specific UGA codons that would otherwise signal translation termination. A specialized set of factors are known to be required for Sec incorporation: a specialized elongation factor that delivers the Sec- tRNASec to the ribosome and unique RNA binding proteins that bind to a Sec insertion sequence (SECIS) in selenoprotein mRNA 3' UTRs. This SECIS-protein complex signals the ribosome to incorporate Sec instead of translation termination. Our prior work has provided molecular characterization of each of the required factors, but the mechanism by which they interact with each other and other cellular components to allow Sec incorporation remains unknown. We provide preliminary evidence that the processive incorporation of 10 Sec residues into the selenium transport protein Selenoprotein P (SELENOP) requires a unique mechanism and additional factors. The overall goals for this proposal are to determine the mechanism by which SECIS binding proteins promote single and multiple Sec incorporation events. We propose to marry our vast library of molecular tools from prior work with new model systems including the first use of a genetically modified zebrafish system to study Sec incorporation as well as CRISPR/Cas9 edited cell lines. All vertebrates possess two SECIS binding proteins encoded by separate genes: SECISBP2 (SBP2) and SECISBP2L. While the mechanism of action for SBP2 is coming into focus, the role for SECISBP2L in Sec incorporation has not been deciphered. Our preliminary data shows that SECISBP2L is essential for the production of a specific 50 kDa selenoprotein, likely SEPHS2, during early development. As such, we have established three model systems to study the synthesis of SELENOP: in vitro translation, expression in transfected mammalian cells and a zebrafish system that will allow unprecedented access to the role of selenoprotein function during development. These are also leveraged and combined with structural biology and transcriptomics to determine how synthesis of the entire selenoproteome is regulated by SECIS binding proteins. In this proposal we propose to 1) Decipher the mechanism by which SECIS elements and SECIS binding proteins enable processive Sec incorporation into the selenium transport protein, SELENOP; 2) Determine the mechanism of SBP2-independent Sec incorporation and the role of SECIS binding proteins in responding to cellular stress; 3) Utilize a zebrafish model system to determine the function of SECISBP2L and the regulation of selenoprotein synthesis by oxidative stress. The successful completion of these aims will bring us significantly closer to our long term goal of developing reagents that will permit selective activation or inhibition of selenoprotein synthesis in vivo.